Development of Transient Recombinant Expression and Affinity Chromatography Systems for Human Fibrinogen |
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Authors: | Grega Popovic Nicholas C. Kirby Taylor C. Dement Kristine M. Peterson Caroline E. Daub Heather A. Belcher Martin Guthold Adam R. Offenbacher Nathan E. Hudson |
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Affiliation: | 1.Department of Chemistry, East Carolina University, Greenville, NC 27858, USA; (G.P.); (N.C.K.); (C.E.D.); (A.R.O.);2.Department of Physics, East Carolina University, Greenville, NC 27858, USA; (T.C.D.); (H.A.B.);3.Department of Biological Engineering, Utah State University, Logan, UT 84322, USA;4.Department of Physics, Wake Forest University, Winston Salem, NC 27109, USA; |
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Abstract: | Fibrin forms the structural scaffold of blood clots and has great potential for biomaterial applications. Creating recombinant expression systems of fibrinogen, fibrin’s soluble precursor, would advance the ability to construct mutational libraries that would enable structure–function studies of fibrinogen and expand the utility of fibrin as a biomaterial. Despite these needs, recombinant fibrinogen expression systems, thus far, have relied on the time-consuming creation of stable cell lines. Here we present tests of a transient fibrinogen expression system that can rapidly generate yields of 8–12 mg/L using suspension HEK Expi293TM cells. We report results from two different plasmid systems encoding the fibrinogen cDNAs and two different transfection reagents. In addition, we describe a novel, affinity-based approach to purifying fibrinogen from complex media such as human plasma. We show that using a high-affinity peptide which mimics fibrin’s knob ‘A’ sequence enables the purification of 50–75% of fibrinogen present in plasma. Having robust expression and purification systems of fibrinogen will enable future studies of basic fibrin(ogen) biology, while paving the way for the ubiquitous use of fibrin as a biomaterial. |
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Keywords: | fibrin fibrinogen hemostasis recombinant protein affinity chromatography |
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